The invention relates to cell biology, and more particularly relates to apoptosis. In its most immediate sense, the invention relates to methods for inducing apoptosis in tumor cells in vivo.
It has long been known that cancerous tumors are created by the unregulated growth of undifferentiated tumor cells. Hence, scientists have long sought therapies that would prevent tumor cells from growing.
To do this, scientists have attempted to disrupt the cell cycle of proliferating HL-60 tumor cells by treating them with N-acetyl-leucinyl-leucinyl-norleucinal (LLnL), a peptide aldehyde known to inhibit the function of proteasome. This arrested the HL-60 cells in the G1 phase of the cell cycle, and in turn prevented them from entering the S phase. When such cells were thus prevented from progressing to subsequent stages of the cell cycle, they died (which is a phenomenon known as programmed cell death, or apoptosis).
However, such experiments focus narrowly upon only one aspect of the cell cycle, namely, inhibiting the function of proteasome (which has been shown to be necessary to normal cell-cycle progression). In fact, progression of cells along the cell cycle comes about as a result of a most complex balancing mechanism. At any given point in the cell cycle, the cells are subjected to biological mechanisms that tend to progress them along the cell cycle as well as biological mechanisms that tend to retain them in whatever phase of the cell cycle they are presently in. Whether or not a cell actually progresses from e.g. the G1 phase to the S phase or from the S phase to the G2 phase or from G2 to mitosis (M phase) is determined by a balance of biological forces acting on that cell; if the positive influence of e.g. cyclins (which act to stimulate progression in the cell cycle) predominates over the negative influence of e.g. kinase inhibitors (which act to arrest cells in their existing cell cycle phase), then the cells may enter or leave the S phase; if not, they may not.
The invention proceeds from the realization that existing approaches to promoting apoptosis of tumor cells do not utilize such positive and negative influences concurrently. As a result, such approaches are not as effective as they might otherwise be.
In accordance with the invention, therapeutically effective doses of two substances are administered in vivo in such a manner that their effects are concurrent. The first substance arrests the cells in a particular phase of the cell cycle, and the second substance prevents the cells from progressing beyond that phase. In this way, the above-referenced balancing mechanism is used to most effectively prevent tumor cells from growing and to thereby induce apoptosis.
In accordance with preferred embodiments, the first substance inhibits intracellular proteasome function, or inhibits activation of NFkB, or both. (If the function of proteasome within the cell is inhibited, activation of NFkB within the cell will likewise be inhibited. This is because NFkB is a heterodimer that has as one of its components a protein known as p50. Normally, the p50 protein is produced by proteolytic processing of its precursor protein p105, which processing is a function of proteasome. When proteasome function is inhibited, the p50 protein is not produced, and instead of producing an active NFkB heterodimer from p50 protein and another protein known as p65 protein, an inactive heterodimer is produced from p105 protein and p65 protein. Additionally, when proteasome function is inhibited, this prevents proteolytic degradation of the IkB protein, which inhibits the function of NFkB by complexing with it and preventing it from entering the nucleus of the cell. In other words, inhibition of proteasome function also inhibits the function of NFkB by reducing the extent to which an inhibitor of NFkB is destroyed.) Two peptide aldehydes, namely LLnL and N-acetyl-leucinyl-valinyl-phenylalaninal (LVP), are suitable for this.
The phase in which inhibition of intracellular proteasome function arrests the cell cycle may vary. As stated above, in HL-60 cells the arrest occurs in the G1 phase, but in DU-145 human prostate carcinoma cells, the arrest occurs in the G2 phase.
In accordance with the preferred embodiment, the second substance inhibits intracellular protein synthesis, or increases intracellular expression of a cyclin-dependent kinase (CDK) inhibitor such as p27KIP1. Ranpirnase is suitable for this. (Ranpirnase is the USAN-approved generic name of the pharmaceutical that is described and claimed in U.S. Pat. No. 5,559,212 and that is presently known by the registered trademark ONCONASE.)
The above-referenced concurrent effects can be achieved by administering both substances separately (as by injections, oral administrations, or both) as long as the substances are administered in such a manner as to cause their effects to be concurrent. It is believed that the above-referenced concurrent effects can also be achieved by administering both substances simultaneously (as in a mixture or other form containing both).